Summary In the presence of anisotropic biochemical or topographical patterns, cells tend to align in the direction of these cues—a widely reported phenomenon known as “contact guidance.” To investigate the origins of contact guidance, here, we created substrates micropatterned with parallel lines of fibronectin with dimensions spanning multiple orders of magnitude. Quantitative morphometric analysis of our experimental data reveals two regimes of contact guidance governed by the length scale of the cues that cannot be explained by enforced alignment of focal adhesions. Adopting computational simulations of cell remodeling on inhomogeneous substrates based on a statistical mechanics framework for living cells, we show that contact guidance emerges from anisotropic cell shape fluctuation and “gap avoidance,” i.e., the energetic penalty of cell adhesions on non-adhesive gaps. Our findings therefore point to general biophysical mechanisms underlying cellular contact guidance, without the necessity of invoking specific molecular pathways.

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The myocardium is a mechanically active tissue typified by anisotropy of the resident cells [cardiomyocytes (CMs) and cardiac fibroblasts (cFBs)] and the extracellular matrix (ECM). Upon ischemic injury, the anisotropic tissue is replaced by disorganized scar tissue, resulting in loss of coordinated contraction. Efforts to re-establish tissue anisotropy in the injured myocardium are hampered by a lack of understanding of how CM and/or cFB structural organization is affected by the two major physical cues inherent in the myocardium: ECM organization and cyclic mechanical strain. Herein, we investigate the singular and combined effect of ECM (dis)organization and cyclic strain in a two-dimensional human in vitro co-culture model of the myocardial microenvironment. We show that (an)isotropic ECM protein patterning can guide the orientation of CMs and cFBs, both in mono- and co-culture. Subsequent application of uniaxial cyclic strain—mimicking the local anisotropic deformation of beating myocardium—causes no effect when applied parallel to the anisotropic ECM. However, when cultured on isotropic substrates, cFBs, but not CMs, orient away from the direction of cyclic uniaxial strain (strain avoidance). In contrast, CMs show strain avoidance via active remodeling of their sarcomeres only when co-cultured with at least 30% cFBs. Paracrine signaling or N-cadherin-mediated communication between CMs and cFBs was no contributing factor. Our findings suggest that the mechanoresponsive cFBs provide structural guidance for CM orientation and elongation. Our study, therefore, highlights a synergistic mechanobiological interplay between CMs and cFBs in shaping tissue organization, which is of relevance for regenerating functionally organized myocardium.

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The human myocardium is a mechanically active tissue typified by the anisotropic organization of cells and extracellular matrix (ECM). Upon injury, the composition of the myocardium changes, resulting in disruption of tissue organization and loss of coordinated contraction. Understanding how anisotropic organization in the adult myocardium is shaped and disrupted by environmental cues is thus critical, not only for unravelling the processes taking place during disease progression, but also for developing regenerative strategies to recover tissue function. Here, we decoupled in vitro the two major physical cues that are inherent in the myocardium: structural ECM and mechanical strain. We show that patterned ECM proteins control the orientation of the two main cell types in the myocardium: human cardiac fibroblasts (cFBs) and cardiomyocytes (hiPSC-CMs), despite their different mechanosensing machinery. Uniaxial cyclic strain, mimicking the local anisotropic deformation of the myocardium, did not affect hiPSC-CMs orientation. It did however induce a reorientation of cFBs, perpendicular to the strain direction, albeit this strain-avoidance response was overruled in the presence of anisotropic structural cues. These findings reveal that the mechanoresponsiveness of cFBs may be a critical handle in controlling myocardial tissue structure and function. To test this, we co-cultured hiPSC-CMs and cFBs in varying cell ratios to reconstruct normal and pathological myocardium. Contrary to the hiPSC-CM monoculture, the co-cultures adopted an anisotropic organization under uniaxial cyclic strain, regardless of the cell ratio. Together, these results identify the cFBs as a therapeutic target to mechanically restore structural organization of the tissue in cardiac regenerative therapies.